For over a decade, ALS-associated SOD1 mutants have been compared to wild-type enzyme, both in vitro and in vivo in an attempt to understand the mechanism(s) of pathogenesis. However, little is known about the molecular mechanisms which underlie pharmacologically-mediated extension of survival in G93A mice. We have identified two chemically distinct (organomanganic) compounds which markedly extend survival in G93A mice when given at disease onset (similar to when human treatment would begin). Based on our results, efforts are underway to conduct human clinical trials in ALS with these compounds. We propose to use these highly efficacious compounds to probe the mechanisms of protection, while continuing our work on the mechanisms of SOD1 mutant-mediated toxicity. We will examine protein expression changes which accompany protection by these compounds and the ultimate loss of protection (Aim 1) in order to gain a better understanding of the mechanisms involved. The magnitude of life extension with these compounds rivals the best yet reported for any compound, even those administered presymptomatically. However, reliable comparisons require comparable treatment regimens. We propose to test compounds that show efficacy when given presymptomatically in an onset administration paradigm (Aim 2) to test the hypothesis that the failure of some human trials may relate to differences in study design. Compelling new evidence suggests that mitochondrial uptake of mutants is critical to disease and that uptake is determined by metallation state (primarily zinc) and the status of an internal disulfide. Herein we offer fundamentally new evidence that a zinc-deficient mutant in vivo directly correlates with disease severity, and that mutant SOD1 (but not wild-type) is covalently cross linked. Aim 3 will identify the cross linked proteins and determine if drug treatment alters it. Aim 4 will extend our studies of in vivo metallation states and thiol status of mutants using enzyme purified from spinal cord as an "in vivo expression system".